As the resolving power of electron microscopes (or other types of microscopes) has improved, efforts to resolve high resolution structures of biological samples have been attempted. However, limitations to high resolution microscopic imaging are often set by the damage a specimen suffers during various sample preparation stages. Many laboratories use ultra-rapid freezing techniques to prepare biological specimens for examination in a microscope to avoid conformational changes of the specimen due to crystallization effects during sample preparation. This requires rapidly freezing a specimen for promoting the formation of amorphous ice, and maintaining the sample at temperatures well below −160° C. to prevent devitrification. Above approximately −160° C., the ice will adopt a crystalline form, which is detrimental to imaging and analysis.
Such frozen specimens require specific microscopes for imaging and analysis. For example, Cryo electron microscopy, or Cryo EM, is a powerful technique for studying frozen hydrated biological specimens in transmission electron microscopes. With respect to biological specimens, as indicated above, the specimen must also be maintained at a low temperature, preferably below −160° C. during the transfer, imaging and analysis process. A Cryo EM includes a specimen holder for mounting the specimen and maintaining it at the required low temperature through the use of a cryogen (or cooling medium) which reduces the temperature of portions of the specimen holder and the specimen itself. This cryogen (such as liquid nitrogen or liquid helium) is stored in an insulated container mounted to one end of the specimen holder, typically identified as a dewar. The dewar is a component of the specimen holder and it comprises an inner vessel enclosed within an evacuated housing. Typically, a dewar has a capacity to hold 0.2-0.5 liters of cryogen and imaging needs to be stopped every 2-3 hours for manual refilling of the dewar. This has many drawbacks, such as limiting the automatic data acquisition time to 2-3 hours, imaging errors because of recalibration required after refilling, manual intervention that leads to inefficiency because a microscope cannot be used when a technician is unavailable, possibility of specimen spoiling during refilling, specimen dislocation, or the like. It is not possible to increase the capacity of a dewar because of the weight restrictions placed on parts of a microscope. Other parts of a microscope such as an anti-contamination device, specimen holder itself, etc. may also require refilling during the operation of the microscope.
This document describes a cryogen refilling system that is directed to solving the issues described above, and/or other problems.